ABSTRACT Unresolving fibrosis due to persistent myofibroblast activation is the distinguishing hallmark of systemic sclerosis (SSc), and accounts for its high mortality. Since current SSc treatments are marginally effective, at best, in reversing fibrosis, there is an urgent need to identify ?druggable? targets. A series of recent findings and genetic studies suggest that Toll-like receptor 4 (TLR4) pathways play key pathogenic roles in SSc by preventing fibrosis resolution. During the previous funding cycle, we showed that TLR4 expression, and levels of endogenous TLR4 ligands, are up- regulated, and TLR4 pathway activity is enhanced, in SSc biopsies despite only sparse inflammation within fibrotic tissues. Additionally, we showed that tenascin-C, a ?damage-associated molecular pattern? (DAMP) produced by activated mesenchymal cells, is persistently elevated in the lesional microenvironment, and co-localizes with TLR4 on tissue myofibroblasts. Remarkably, tenascin-C itself elicits TLR4-mediated myofibroblast activation and related fibrotic responses. The deubiquitinase enzyme A20, previously linked to inflammatory TLR4 signaling and implicated in autoimmune diseases, was recently identified as a candidate gene in SSc. We demonstrated that A20 is expressed in normal and SSc fibroblasts and negatively regulates fibrotic responses. Remarkably, fibrosis was reduced in mice carrying non-functional TLR4 or lacking tenascin-C, and its resolution was accelerated. Our working hypothesis, based on these observations, is that fibrosis chronicity in SSc is maintained by aberrant TLR4 signaling in tissue myofibroblasts responding to DAMPs within their microenvironment, impeding fibrosis resolution and tissue regeneration. Persistent cross-talk of environment with resident fibroblasts resulting from dysregulation of DAMPs and TLR4 pathways therefore represents a perturbation that is fundamental to the pathogenesis of non-resolving fibrosis in SSc. To test our hypothesis, Aim 1 will employ genetic and pharmacological approaches to determine cell- and ligand-specific roles and mechanisms of TLR4/MD2 signaling in fibrosis. Aim 2 will evaluate fibroblast-specific regulation and function of A20 as a novel endogenous regulatory mechanism in the setting of SSc. Aim 3 will determine the site, source, and relation to clinical phenotypes of TLR4 pathway expression in SSc skin biopsies. Employing human samples from the established Northwestern and Yale Scleroderma Programs, combined with cell-based assays, disease models and engineered mice, we will generate new knowledge to advance the understanding of SSc in particular, and fibrosis in general. In turn, this information will guide discovery and application of novel precision therapies that target persistent myofibroblast activation in SSc.